A model for transcription activation has recently been developed based on the structure of E. Coli RNA polymerase, and analysis of activation by the CRP protein. In this model, the activator protein directly contacts the RNA polymerase alpha or subunit, resulting in increased transcription initiation. The long range goal of this research is to understand the molecular mechanisms of transcription activation, especially where multiple activator proteins simultaneously regulate expression of a gene(s). The objective of this proposal is to investigate the details of transcription activation, utilizing the E. coli L-rhamnose rhaBAD genes as a model. Full transcription of rhaBAD requires two activator proteins, RhaS and CRP. The central hypothesis is that while activator-RNA polymerase interactions likely contribute to transcription activation, activator-activator and activator-DNA interactions may also be important. In order to understand how transcription activation occurs, it is essential to determine the nature of the protein-protein and protein-DNA interactions that are required. Three specific aims will be pursued to accomplish the objectives of this proposal: 1) characterize DNA binding by RhaS; 2) characterize transcription activation by RhaS; and 3) characterize transcription activation by CRP. The role of protein-DNA interactions will be investigated by isolating mutations which identify the amino acid-base contacts between RhaS and DNA. To understand the role of protein-protein interactions in activation, mutations will be isolated that identify the specific amino acids in RhaS and CRP that are responsible for transcription activation. Once activation defective mutations are identified in RhaS and CRP, second site suppressors will be isolated. The second site suppressor mutations will identify the protein-protein interactions that are important for transcription activation. The expectation is that this research will identify a variety of interactions that contribute to transcription activation. This work is significant because it will contribute to a broader understanding of the mechanisms used for activation, especially with multiple activators.